FK-506 type macrolide intermediate

ABSTRACT

An intermediate, useful in the conversion of FK-506 to FK-525 and analogous 23-membered ring macrolides, of the structure: ##STR1##

CROSS REFERENCE TO RELATED APPLICATIONS

The instant case is a divisional application of pending U.S. Ser. No.07/374,508, filed Jun. 30, 1989 now U.S. Pat. No. 5,164,525.

BACKGROUND OF THE INVENTION

1) Field of the Invention

This invention relates to a new synthetic process for producingmacrolide immunosuppressant FK-506 type precursor intermediates.

2) Brief Disclosures in the Art

The novel 23-membered tricyclo-macrolide FK-506 isolated andcharacterized by Tanaka, Kuroda, and co-workers, see JACS, 109, pp.5031, 1987, and EPO Publication No. 0,184,162, has been shown to possessexceptional immunosuppressive activity. The potential usefulness of suchan agent in bone marrow and organ transplantations coupled with itsunique structural features has prompted many in the field to initiate aneffort towards the total synthesis of FK-506 (1). ##STR2##

A total synthesis of FK-506 has been achieved by Ichiro Shinkai'sProcess Chemistry Group at Merck & Co., Inc. by R. Volante, D. Askin, etal. as published in J. Am. Chem. Soc., 1989, Vol. 111, 11, p. 1157. Apatent application, U.S. Ser. No. 07/596,847, filed Oct. 12, 1990, beinga continuation of U.S. Ser. No. 07/375,091, filed Jun. 30, 1989, nowabandoned, being a continuation-in-part of U.S. Ser. No. 07/295,877,filed Jan. 11, 1989, now abandoned, claims this synthesis and is herebyincorporated by reference for this particular purpose.

The total synthesis is an extremely elegant and sophisticted work in thefield of macrolide chemistry and requires 54 discrete synthetic steps toFK-506 starting from divinyl carbinol³ and quinic acid⁴. See ³ Askin,D.; Volante, R. P.; Reamer, R. A.; Ryan, K. M.; Shinkai, I., TetrahedronLett., 1988, 29. p. 277, and ⁴ Mills, S.; Desmond, R.; Reamer, R. A.;Volante, R. P.; Shinkai, I., Tetrahedron Lett., 1988, 28, p. 281.

However, to make other potential immunosuppressant derivatives of FK-506via this synthetic scheme would be very laborious and particularly thosederivatives having a different moiety in the C₁ -N₇ position. It wouldbe desirable to possess a short, convenient process to synthesize FK-506type macrolides from a readily obtainable intermediate in high yield.

There are no known degradation routes disclosed in the literature fromFK-506 to a useful synthetic intermediate. In particular, it is notdisclosed how to cleave the C.9, C.10 bond cleanly and in high yield,selectively protect the many alcohol functions and selectively adjustC.10 to the aldehyde oxidation state protected as the labile dimethylacetal. Furthermore, it is not disclosed as to a method by which theC.26-OH could be deacylated, since all attempts to deacylate C.26-OHwith C.22 as the ketone leads to decomposition via a retroaldolfragmentation. See Tanaka, H.; Kuroda, A.; Marusawa, H.; Hatanaka, H.;Kino, T.; Goto, T.; Hashimoto, M.; Taga, T., J. Am. Chem. Soc., 1987,109, p. 5031.

What is needed in the art is a convenient, relatively low multistepsynthesis of an FK-506 degradation intermediate to synthesize otherFK-506 type macrolides having different C₁ -N₇ moieties.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates structure XVI and

FIG. 2 illustrates structures I, XV, XVII, XVIII, and the values of Rfor each structure.

SUMMARY OF THE INVENTION

We have discovered a short, ten step convenient route to the versatileintermediate 11, starting with FK-506, as depicted below, which can beused to produce other C₁ -N₇ FK-506 type macrolides, including FK-525.The overall yield from the starting material (FK-506) to 11 is 12%,which means that only 5-6 g of FK-506 is necessary to produce 1 gram of11.

This route has the ability to prepare quantities of about 1 g of theintermediate 11 in a laboratory setting without the use of special largescale apparatus. By contrast, a period of several months is required forthe synthesis of 1 g of 11 based on the above-described total synthesis.

In accordance with the invention, there is provided a compound of theformula: ##STR3## wherein P/P¹ /P² are independently defined as H ortri(hydrocarbo)silyl, wherein said hydrocarbo groups are independentlychosen from C₁ -C₄ linear or branched alkyl, phenyl or benzyl, such thatP can be selectively removed in the presence of P¹ /P² and R is selectedfrom allyl, propyl, ethyl or methyl, and R₁ is methyl or ethyl.

Further provided is a process for degrading an FK-506 type macrolide toa useful intermediate therefor comprising the steps of: ##STR4##

a) contacting I, where R is selected from allyl, methyl, ethyl orpropyl, with a silylating agent in the presence of an amine hydrogenacceptor to form II; ##STR5##

b) contacting II with Pb(OAc)₄ in a dry inert organic solvent containingmethanol at a temperature in the range of 10°-40° C. to form III;##STR6##

c) contacting III with a silylating agent at 0°-10° C. in the presenceof an organic amine hydrogen acceptor to form V; ##STR7##

d) contacting V with LiAlH₄ at 0°-5° C. for a sufficient time to formthe triols VI (R/S); ##STR8##

e) contacting the triols VI (R/S) with silylating agent at -10° to 0° C.for a sufficient time to form the alcohol VII (R/S); ##STR9##

f) contacting alcohol VII (R/S) with silylating agent in the presence ofan organic amine hydrogen acceptor at a temperature in the range of0°-10° C. for a sufficient time to form the ether VIII (R/S); ##STR10##

g) contacting VIII with aqueous acetic acid at 25°-50° C. to form theprimary alcohols IX, 22(S) and 22(R); ##STR11##

h) contacting IX with oxalyl chloride, DMSO and an organic amine at atemperature in the range of about -80° to -60° C. or alternatively CrO₃-pyridine complex at about 25° C. in a dry inert organic solvent for asufficient time to form X; ##STR12##

i) contacting X with methanol, trialkylorthoformate and pyridiniump-toluenesulfonate at 0°-10° C. in a dry organic solvent for asufficient time to form XI; ##STR13## wherein P/P¹ /P² are independentlydefined as H or tri(hydrocarbo)silyl, and P_(a) and P_(b) are either Hor P, and wherein said hydrocarbo groups are independently chosen fromC₁ -C₄ linear or branched alkyl, phenyl or benzyl, such that P can beselectively removed in the presence of P¹, P², or both, and R₁ is methylor ethyl.

BRIEF DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The invention described herein concerns the synthesis of a versatilesynthetic intermediate 11 for the preparation of FK-506 type macrolidesand may be readily understood by reference to generalized Flow Charts Aand B, and the specific process for FK-506 as depicted in Flow Charts A¹and B¹.

This process degrades an FK type macrolide, i.e., FK-506, in ten stepsto a C.10-C.34 fragment with the C.10 position at the aldehyde oxidationlevel protected as a dialkylacetal, e.g. dimethylacetal, and the C.26hydroxyl group selectively exposed for acylation with an appropriateN-protected, secondary amino-acid, e.g. N-t-Boc-L-proline. ##STR14##

The starting FK-506 and corresponding methyl (FK-523), ethyl (FK-520),propyl, and FK-525 (proline) derivatives are known in the art and aredescribed in Fujisawa's EPO Publication No. 0,184,162, supra.

As is seen in Flow Chart A, the initial bis-(trialkylsilylation) ofFK-506 type macrolide (I) affords II.

Generally, this step is conducted under anhydrous conditions, e.g. drynitrogen and a dry inert organic solvent, e.g. dichloromethane,diethylether, dioxane, benzene, toluene, or xylene, in the temperaturerange of -10° to +25° C., and the presence of an organic nitrogen protonacceptor, e.g. 2,6-lutidine for a sufficient time to produce thebis-protected product II. Time required generally in the range of 1 to24 hours. The protecting agent is a triorganosilylating agent for freehydroxyl groups, e.g. triisopropylsilyltriflate (TIPSOTf), preferablybeing a reagent that provides the triisopropylsilyl group, since it isdesired that the C-24 and C-32 hydroxyl groups be protected with themost difficult to remove silyl protecting groups in order that they beremoved last.

The bis-protection step is performed with a conventionaltri(hydrocarbo)silyl protecting groups P, P¹ and P² .

P, P¹ and P² are selected from conventional trihydrocarbosilylprotecting groups, wherein P can be selectively removed in the presenceof P¹ and P² ; P¹ can be selectively removed in the presence of P², byappropriate choice of deprotection reaction conditions as is standard inthe art (e.g. choice of reaction temperature, reaction solvent, reagentconcentration, reaction time, or strength of reagent in cases of acidcatalyzed hydrolysis.

The protecting groups P, P¹ and P² can be conventional in the art andare tri(hydrocarbo)silyl, wherein said hydrocarbo group is independentlychosen from C₁ -C₄ linear or branched alkyl, phenyl or benzyl, includingmixtures thereof.

Representative examples of tri(hydrocarbo)silyl groups includetrimethylsilyl- (TMS), triethylsilyl- (TES) as P;isopropyldimethylsilyl-, t-butyldimethylsilyl- (TBS),triisopropylsilyl(TIPS), triphenylsilyl-, tribenzylsilyl- as P² ;phenyldimethylsilyl-, benzyldimethylsilyl-, diethylisopropylsilyl- asP¹, and the like.

Preferred is where P² is triisopropyl (TIPS), P¹ is t-butyldimethylsilyl(TBS) and P is triethylsilyl (TES).

Methods of deprotection are standard in the art and include: (a) removalof P groups, e.g. TES, by acetic acid/water/tetrahydrofuran at 40° C.for about 12 hours; (b) removal of P¹ groups, e.g. TBS, by treating withanhydrous tetrabutylammonium fluoride in THF at 0°-25° C.; (c) removalof P² groups, e.g. TIPS, by treating with acetonitrile/50% aqueous HF at0°-10° C.

The C.9-C.10 bond of II is next cleaved in high yield with leadtetraacetate in an inert solvent mixture, e.g., methanolic benzene, toafford a mixture of the methyl ester III and the correspondingvalerolactone IV.

This oxidation step is generally carried out with Pb(OAc)₄ as theoxidizing agent in a dry inert solvent, e.g. C₆ -C₈ aromatichydrocarbon, i.e., benzene, toluene, xylene or a C₁ -C₄ alcohol, i.e.,methanol, ethanol and the like, at a temperature of about 10°-40° C.,preferably 25° C. for a sufficient time to produce III. Varying amountsof valerolactone IV will be formed which can be converted to III bytreating with methanol/anhydrous K₂ CO₃ for a sufficient time tosubstantially convert IV to III.

The free hydroxyl group in III is next protected by P¹ producing V, aprotecting group more easily removable than P². Preferred in thiscontext is the t-butyldimethyl group (TBS) which can be incorporated bytreating III in a dry inert organic solvent, e.g. C₆ -C₈ aromatic C₂ -C₆ethers or halogenated C₁ -C₄ alkane, and the like, i.e. dichloromethane,with a silylating agent, e.g. TBS triflate and an organic amine protonacceptor, i.e. 2,6-lutidine, at 0°-25° C. for a sufficient time tosubstantially silylate the free hydroxy group.

Exhaustive hydride reduction of V affords the triols VI (R,S) as, e.g.,a 2.5 to 1 mixture of inseparable C.22 epimers with the (R)-epimerpredominating. Although only the major (R)-C.22 epimer has been thus farused for the synthesis of FK-506 and congeners, it is reasonablybelieved that the (S)-C.22 epimer is also useful.

The reduction step is carried out with a strong reducing agent,preferably LiAlH₄, in a dry inert organic solvent, e.g. C₂ -C₄ ether,i.e. tetrahydrofuran, diethylether, and the like, at 0°-25° C., under adry atmosphere for a sufficient time to substantially reduce the ketoneto the secondary alcohol, reduce the ester group to an alcohol andcleave the pipecolinic ester to afford VI.

In a key step, selective protection of the C.10 and C.26 alcohols isachieved with, e.g., TESCl/pyridine to afford the bis-protected, e.g.,bis(triethylsilyl ethers) VII (R,S).

VI is then treated with this protecting group P, being more easilyremovable than P² and P¹, and preferably is triethylsilyl. Theconditions generally include treating VI with a silylating agent, e.g.triethylsilylchloride, in an anhydrous, inert solvent, e.g.dichloromethane, diethylether, tetrahydrofuran, in the presence of anamine nitrogen proton acceptor, e.g. 2,6-lutidine, triethylamine, or asolvent such as pyridine can be used accomplishing both goals. Thetemperature is generally in the range of -10° to 25° C. to achievesubstantial yields of VII.

The exposed C.22-OH (R and S epimers) is then silyl protected as, forexample, the tertbutyldimethylsilyl ether, as described above, to affordVIII (R,S).

Selective deblocking of the terminal primary C.10-silylated ether thengives the chromatographically separable C.22-epimers IX (R) and (S).

The deblocking is carried out in a weak acid mixture, e.g. HOAc/THF/H₂O, at 25°-50° C., preferably 40° C., to produce a substantial yield ofthe IX (R, S) alcohols.

IX is subjected to Swern oxidizing conditions involving oxalyl chloride,dimethylsulfoxide, and triethylamine, at -78° to -40° C. or CrO₃-pyridine at 25° C. in a dry inert solvent, e.g. dichloromethane, for asufficient time to obtain a substantial yield of the aldehyde X.

X is converted to the dimethylacetal and the ether group at position 26is deprotected by treatment with triethylorthoformate and a weak acidcatalyst, pyridinium p-toluenesulfonate, in a dry inert organic solvent,e.g. THF, methanol, benzene, CH₂ Cl₂, and the like, at 0°-25° C., for asufficient time to substantially form the key intermediate XI. Note thattriethylorthoformate can also be used in place of the trimethyl analogand that the subsequent triethyl analog is also deemed to be coveredwithin the scope of this invention.

XI can be reacted with an N-protected secondary amino acid, e.g.N-t-Boc-L-proline, under the conditions involving the reagentdicyclocarbodiimide and N,N-diethylaminopyridine in an inert dry organicsolvent, e.g. dichloromethane at -50° to -25° C., for a sufficient timeto form the condensation product, e.g. XIV.

For example, the C.26 alcohol of 11 can be acylated (as designated byP_(b)) with a variety of N-protected amino acids such asN-(tert-butoxycarbonyl)-(S)- pipecolic acid with1,3-dicyclohexylcarbodiimide/dimethylaminopyridine to afford 12, anintermediate in the total synthesis of FK-506. See Jones, T. K.; Mills,S. G.; Reamer, R. A.; Askin, D.; Desmond, R.; Volante, R. P.; Shinkai,I., J. Am. Chem. Soc., 1989, 111. p. 1157.

Other secondary amino acids (e.g. in their N-protected form, e.g.N-t-Boc) can also be employed in the synthesis starting with thecompound 11 condensation step and includes all naturally occurring aminoacids and those known synthetic variations in the art which includethose of the following formulae:

Acyclic Secondary Amino Acids ##STR15## where R_(a) =C₁ -C₁₀ alkyl,aryl, or substituted alkyl; R_(b) =H, C₁ -C₁₀ alkyl, aryl, orsubstituted alkyl; and R_(c) =H, C₁ -C₁₀ alkyl, aryl, or substitutedalkyl, said substituents, including halo, C₁ -C₄ alkoxy, e.g. chloro,methoxy, and the like.

Representative examples include N-methyl, N-ethyl, N-benzyl, N-phenylsubstituted L- and D-forms (and racemates) of alanine, valine, leucine,isoleucine, phenylalanine, tyrosine, diiodotyrosine, thyroxine, serine,threonine, methionine, cysteine, cystine, aspartic acid, glutamic acid,lysine, arginine, known synthetic variants thereof, sarcosine, and thelike;

Cyclic Secondary Amino Acids ##STR16## where R_(d) =H, C₁ -C₁₀ alkyl,aryl or branched alkyl, which may be in the alpha or beta configuration;R_(e) and R_(f) are carbon-containing chains joined with NH to form a4-10 membered carbon-nitrogen ring, which can be saturated, unsaturatedor partially unsaturated, can contain one or more O, S or N heteroatomsand which can be ring substituted.

The above secondary amino acids are converted to their N-t-Boc protectedform, by conventional procedure, and can be utilized in the place ofsarcosine, proline, or pipecolinic acid.

Representative examples include L- and D-forms of proline,hydroxyproline, N-methyltryptophan, N-methylhistidine, 2-pipecolinicacid, known synthetic variants thereof, and the like, wherein saidsubstituents include halo, C₁ -C₄ alkoxy, i.e. chloro, methoxy, and thelike.

Also described is the synthetic sequence for converting the condensationproduct to the corresponding FK type macrolide. For example, 14 isconverted to FK-525.

The above procedure is applicable to all of the FK-506 macrolide family,e.g. FK-506 (R¹ =allyl), FK-525 (R¹ =allyl), FK-520 (R¹ =ethyl), FK-523(R¹ =methyl) and the FK-506 analog where R¹ =propyl.

The following examples are illustrative of carrying out the instantinvention and should not be construed as being a limitation on the scopeor spirit of the invention.

EXAMPLE 1 ##STR17##

24-32-FK-506-bis-(triisopropylsilyl)-ether 2

To a 0° C. solution of FK-506 1 (10.2 g, 12.68 mmol) in 125 mL sievedried CH₂ Cl₂ was added 2,6-lutidine (7.4 mL, 63.5 mmol) and TIPSOTf(14.3 mL, 53.2 mmol). After stirring at 0° C. for 1.5 h, the yellowtinted solution was warmed to 25° C. and aged for 16 h. The mixture wasthen cooled to 0° C. and methanol (1.55 mL, 38.2 mmol) was addeddropwise and the resulting solution was aged for 15 min. The mixture waspartitioned with sat'd aqueous NaHCO₃ (500 mL) and CH₂ Cl₂ (200 mL) andthe layers were separated. The aqueous phase was reextracted with CH₂Cl₂ (2×200 mL). The combined CH₂ Cl₂ phase was washed with water (200mL), dried with MgSO₄ and concentrated in vacuo to afford 20 g of ayellow viscous oil. Chromatography on silica gel (325 g, 230-400 mesh)with hexanes/ethyl acetate (5:1) as the eluent afforded 14.04 g (99%) of2 as a colorless foam which was characterized by ¹ H, ¹³ C NMR, IR andMS. Anal. Calcd for C₆₂ H₁₀₉ NO₁₂ Si₂ : C, 66.685; H, 9.838; N, 1.25.Found: C, 66.88; H, 9.71; N, 1.25.

EXAMPLE 2 ##STR18## Hydroxy-ester 3

To a 25° C. solution of 24,32-FK-506-bis(triisopropylsilyl) ether 2(23.1 g, 20.7 mmol) in 300 mL sieve dried benzene and 100 mL sieve driedmethanol was added Pb(OAc)₄ (9.67 g, 21.8 mmol) and the resultingmixture was aged at 25° C. for 4 h. The mixture was then quenched intosat'd aqueous NaHCO₃ (800 mL) and extracted with ethyl acetate (3×350mL). The combined ethyl acetate phase was washed with water (350 mL),dried with MgSO₄ and concentrated in vacuo to afford 22.9 g of a whitegummy foam. The foam was dissolved in methanol (300 mL) at 25° C.concentrated in vacuo to remove any residual ethyl acetate. The foam wasthen redissolved in sieve dried methanol (350 mL) at 25° C. andanhydrous K₂ CO₃ (145 mg) was added. After 2 h additional K₂ CO₃ (43 mg)was added. After 5 h total reaction time at 25° C. the solution wasdecanted away from the solid K₂ CO₃ and was concentrated in vacuo toafford 23.4 g of a yellow tinted gum. Chromatography on silica gel (1150g, 230-400 mesh) with CH₂ Cl₂ /acetone (20:1) as the eluent followed byCH₂ Cl₂ /acetone (15:1) afforded 2.52 g (10.6%) of the valerolactone 4as a colorless foam and 16.8 g (69%) of the desired methyl ester 3 as acolorless foam.

EXAMPLE 3 ##STR19## C.14 TBS-ether 5

To a 0° C. solution of the hydroxy-ester 3 (16.8 g, 14.25 mmol) in 210mL dry CH₂ Cl₂ was added 2,6-lutidine (3.3 mL, 28.3 mmol) and thentert-butyldimethylsilyl triflate (4.9 mL, 21.3 mmol) and the solutionwas stirred at 0° C. for 2 h, then it was allowed to warm to 25° C.After 1 hr at 25° C additional tert-butyldimethylsilyl triflate (0.35mL, 1.52 mmol) was added to the 25° C. solution. After an additional 1.5hr age, the solution was cooled to 0° C. and sieve dried methanol (0.58mL, 14.3 mmol) was added and the mixture was aged at 0° C. for 15 min.The mixture was partitioned with saturated aqueous NaHC0₃ (1000 mL) andextracted with CH₂ Cl₂ (3×300 mL). The combined CH₂ Cl₂ layer was washedwith water (1×300 mL), dried with MgSO₄ and concentrated in vacuo toafford 21.4 g of a tan oil that was purified by chromatography on silicagel (1,070 g, 230-400 mesh) with hexanes/ethyl acetate (5:1) as theeluent. The TBS ether 5 (14.4 g, 78.3%) was isolated as a white gummyfoam and gave satisfactory ¹ H and ¹³ C NMR, IR and mass spectral data.Anal. Calcd for C₇₀ H₁₂₉ NO₁₄ Si₃ : C. 65.02; H, 10.055; N, 1.08. Found:C, 65.I8; H, 10.06; N, 1.09.

EXAMPLE 4 ##STR20## Triols 6 (R/S)

To a 0° C. solution of methyl ester 5 (13.3 g, 10.28 mmol) in sievedried THF (375 mL) was added lithium aluminum hydride (1.18 g, 31.1mmol) in several portions, and the gray suspension was aged at 0° C. for3 h. Diethyl ether (300 mL) was added and then water was cautiouslyadded dropwise until bubbling had stopped (several mL). The mixture wasthen partitioned with saturated Na₂ SO₄ (400 mL) and extracted withethyl acetate (3×400 mL). The combined ethyl acetate layer was washedwith brine (400 mL), dried with MgSO₄ and concentrated in vacuo toafford 11.8 g of a brownish gummy foam. The crude foam was purified bychromatography on silica gel (580 g, 230-400 mesh) eluting withhexanes/ethyl acetate (3:1) to afford 5.9 g (65.6%) of the triols 6 asan inseparable 2.5 to 1 mixture of C.22-R/S epimers, respectively. Anal.Calcd for C₆₀ H₁₂₀ Si₃ O₉ : C, 67.36; H, 11.305. Found: C, 67.39; H,11.38.

EXAMPLE 5 ##STR21## C.22 alcohols 7(R/S)

To a -10° C. solution of triols 6(R/S) (2.33 g, 2.18 mmol) in sievedried pyridine (33 mL) was added TESCl (0.740 mL, 4.40 mmol) over a 5minute period. After a 1.75 h age at -10° C., additional TESCl (0.092mL, 0.55 mmol) was added. After an additional 1 h age, anhydrousmethanol (0.088 mL, 2.17 mmol) was added and the mixture was aged at-10° C. for 0.25 hr. The mixture was partitioned with saturated aqueousNaHC0₃ (100 mL) and extracted with CH₂ Cl₂ (3×100 mL). The combined CH₂Cl₂ layer was washed with saturated aqueous NaHC0₃ (100 mL), dried withMgSO₄ and concentrated in vacuo to afford 3.7 g of a tan viscous oil.The crude mixture was purified by chromatography on silica gel (175 g,230-400 mesh) with hexanes/ethyl acetate (18:1 to 15:1) as the eluent toafford 2.726 g (96.3%) of the C.22 alcohols 7(R/S) as a colorless foam.Anal. Calcd for C₇₂ H₁₄₈ O₉ Si₅ : C, 66.605; H, 11.49. Found: C, 66.82;H, 11.77.

EXAMPLE 6 ##STR22## C.22-TBS ethers 8(R/S)

To a 0° C. solution of C.22 alcohols 7(R/S) (4.55 g, 3.50 mmol) in 100mL sieve dried CH₂ Cl₂ was added 2,6-lutidine (1.20 mL, 10.3 mmol) andthen tertbutyldimethylsilyl triflate (1.60 mL, 6.97 mmol). After 0.5 hthe mixture was brought to 25° C. and aged for 14 h. The mixture wascooled to 0° C. and anhydrous methanol (215 microliters, 5.30 mmol) wasadded and the mixture was aged for 0.5 h. The mixture was partitionedwith 50% saturated aqueous NaHC0₃ (125 mL) and extracted with CH₂ Cl₂(3×125 mL). The combined CH₂ Cl₂ layer was washed with water (125 mL),dried with magnesium sulfate and concentrated in vacuo to afford 5.3 gof a tan oil. The crude mixture was purified by chromatography on silicagel (260 g, 230-400 mesh) eluting with hexanes/ethyl acetate (15:1) toafford 4.86 g (98%) of the C.22 TBS ethers 8(R/S) as a gummy white foam.Anal. Calcd for C.sub. 78 H₁₆₂ O₉ Si₆ : C, 66.32, H, 11.56. Found: C,65.93; H, 11.77.

EXAMPLE 7 ##STR23## 22(R) and 22(S)-C.10 primary alcohols 9

To a solution of bis-TES ethers 8(R/S) (4.85 g, 3.43 mmol) in 155 mL THFwas added 15.5 mL water and 30 mL of acetic acid over a 10 minuteperiod. The mixture was heated to 40° C. and aged for 12 h, then at 50°C. for 2.5 hr. The mixture was cooled to 0° C. and poured slowly into asolution/suspension of 72 g NaHCO₃ in 450 mL H₂ O. The mixture was thenextracted with ethyl acetate (3×450 mL) and the combined ethyl acetatelayer was washed with saturated aqueous NaHCO₃ (115 mL), brine (115 mL)and dried with magnesium sulfate. The volatiles were removed in vacuo toafford 5.01 g of a tan gum that was purified by silica gelchromatography (675 g, 230-400 mesh). Gradient elution withhexanes/ethyl acetate (8:1) to (2:1) afforded 742 mg (16.6%) of pureless polar 22(S)-C.10 primary alcohol 9(S), 305 mg (6.8%) of mixedfractions and 2.315 g (52%) of the 22(R)-C.10 primary alcohol 9(R) whichhad ¹ H and ¹³ C NMR, IR and MS data consistent with the structure.Anal. Calcd for C₇₂ H₁₄₈ O₉ Si₅ : C, 66.605; H, 11.489. Found: C, 66.47;H, 11.73.

EXAMPLE 8 ##STR24## Aldehyde 10

To a -78° C. solution of oxalyl chloride (148 microliters, 1.70 mmol) in10 mL sieve dried CH₂ Cl₂ was added a solution of dimethyl sulfoxide(200 @l, 2.82 mmol) in 4 mL CH₂ Cl₂ over a period of 5 min and theresulting mixture was aged at -78° C. for 0.5 h. A solution of theprimary alcohol 9(R) (1.06 g, 0.816 mmol) in 10 mL CH₂ Cl₂ was added tothe -78° C. chlorosulfonium salt solution followed by a 5 mL CH₂ Cl₂flush. The resulting white slurry was aged at -78° C. for 1.5 h, thentriethylamine (983 microliters, 7.05 mmol) was added and the solutionwarmed to -40° C. and aged at -40° C. for 1 h. Aqueous NaHS0₄ (0.5M, 75ml) was added at -40° C. and the mixture was extracted with hexanes(4×100 mL). The combined hexane layer was washed with water (1×50 ml),dried with MgSO₄ and concentrated in vacuo to afford 1.05 g of crudematerial that was chromatographed on silica gel (90 g, 230-400 mesh).Elution with hexanes/ethyl acetate (15:1) gave 977 mg (95.8%) of thealdehyde 10 as a white foam. Aldehyde 10 gave ¹ H and ¹³ C NMR, IR andMS data in accord with its structure.

EXAMPLE 9 ##STR25## Dimethyl acetal 11

To a solution of aldehyde 10 (2.07 g, 1.60 mmol) at 0° C. in 105 mLsieve dried THF was added methanol (155 mL), trimethylorthoformate (3.13mL, 28.6 mmol) and pyridinium para-toluenesulfonate (555 mg, 2.2 mmol)and the mixture was warmed to 18° C. After 2 h, 444 mg of pyridiniumpara-toluenesulfonate was added, and the mixture was warmed to 25° C.After 3 h at 25° C., pyridine (4.9 mL, 60.5 mmol) was added with icebath cooling and the mixture was poured into 250 mL saturated aqueousNaHCO₃ and extracted with CH₂ Cl₂ (3×200 mL). The combined CH₂ Cl₂ layerwas washed with 50% aqueous NaHCO₃ (120 mL), dried with MgSO₄ andconcentrated in vacuo. The resulting crude oil was chromatographed on200 g SiO₂ (230-400 mesh) eluting with hexanes/ethyl acetate ((15:1),1.6 L; (8:1), 850 mL; (3:1), flush) to afford 1.637 g (83.5%) of thedimethyl acetal 11 as a colorless oil, which exhibited ¹ H and ¹³ C NMR,IR and MS consistent with its structure. Anal. Calcd for C₆₈ H₁₃₈ O₁₀Si₄ : C, 66.501; H, 11.325. Found: C, 66.43;, H, 11.68.

EXAMPLE 10 ##STR26## N-BOC proline ester 14

To a solution of C.26 alcohol 11 (388.5 mg, 0.316 mmol) in 6 mL dry CH₂Cl₂ at -50° C. was added solid N-Boc-(L)-Proline (273 mg, 1.27 mmol)then 1,3-dicyclohexylcarbodiimide (DCC, 262 mg, 1.27 mmol) andN,N-dimethylaminopyridine (DMAP, 7.8 mg, 0.064 mmol) and the resultingsolution was aged at -50° C. for 1.5 h. The mixture was warmed to -19°C. and aged for 30 h, then N-BOC-(L)-proline (136 mg), 131 mg DCC and 4mg DMAP were added and the slurry was aged again for 24 h. The slurrywas then warmed to 25° C., filtered to remove the precipitate and thefilter cake was washed with hexanes/ethyl acetate (6:1). The volatileswere removed in vacuo to afford 970 mg of a crude oil that was purifiedby silica gel chromatography (95 g, 230-400 mesh). Elution withhexanes:ethyl acetate (3:1) gave 435.8 mg (96.7%) of the N-BOC prolineester 14. The ester 14 exhibited ¹ H and ¹³ C NMR, IR and MS data inaccord with its structure. Anal. Calc'd for C₇₈ H₁₅₃ Si₄ O₁₃ N: C,65.726; H, 10.819; N, 0.983. Found: C, 65.89; H, 11.10; N, 1.00. Thematerial had a rotation [α]²⁵ =-42.73° in methylene chloride at c=1.16g/100 ml.

Subjecting 14 to the following synthetic steps, as also outlined incompanion copending case Ser. No. 07/375,091 in favor of U.S. Ser. No.07/596,847, filed Oct. 12, 1990, filed Jun. 30, 1989, now abandoned,will result in regenerating the FK-macrolide, specifically, for example,the immunosuppressant FK-525.

EXAMPLE 11(2R,4S,5R,6S,8S,12R,13R,15S,16R,17S,1'R,3'R-4'R)-E,E-4,6-dimethoxy-2,8,10,16,18-pentamethyl-5,13-bis-t-butyldimethylsilyloxy-12-(prop-2'-en-1'-yl)-15-triisopropylsilyloxy-17-((N-t-butylcarboyloxy-L-prolinoyl)-19-(3'-methoxy-4'-triisopropylsilylyoxycyclohexyl)-nonadecan-10,18-dienal,15 ##STR27##

Dimethyl acetal 14 (427 mg, 0.299 mmol) was dissolved in 13 ml ofmethylene chloride under a nitrogen atmosphere and glyoxylic acidmonohydrate (276 mg, 3 mmol) and acetic acid (171 microliters, 10 equiv)were added. The resulting solution was stirred at 40° C. for 2.5 hr. Themixture was cooled to 25° C. and poured into 25 ml of saturated sodiumbicarbonate solution at 0° C. The phases were separated and the aqueousphase was extracted with 3×75 ml of methylene chloride. The organicphases were combined, dried over sodium sulfate, and concentrated invacuo. The resulting material was purified by column chromatography on50 g of silica gel eluting with hexanes:ethyl acetate (7:1) to give thedesired aldehyde 15 (390 mg, 95%). The material was homogeneous by both¹ H and ¹³ C NMR.

EXAMPLE 12 Preparation of 2-P-Methoxybenzyl-Acetic Acid Phenyl AlanineDerived Oxazolidinone Imide, 19 ##STR28## A. Preparation of 16

p-Methoxybenzyl alcohol (83.1 g) was dissolved in 75 ml of toluene andadded dropwise (under a nitrogen atmosphere) over a period of 30 min. tosuspension of sodium hydride (53 g of a 60% oil dispersion, 2.20equivalents) in 300 ml of toluene. The internal temperature rose from24° C. to 35° C. during the addition. After hydrogen evolution hadceased (ca. 20 min.), 2-bromoacetic acid (in 400 ml of toluene) wasadded over 1 hour dropwise under a nitrogen atmosphere keeping theinternal temperature below or equal to 40° C. The addition of2-bromoacetic acid was highly exothermic and external cooling wasnecessary. After 45 min., the mixture was diluted with 400 ml of tolueneand heated at 95° C. for 2 hr. The mixture was cooled to 25° C. andquenched by the addition of 400 ml of water and the layers wereseparated. The aqueous phase was extracted with 2×200 ml ofmethyl-t-butyl ether. The aqueous layer was acidified with 60 ml of 1NH₂ SO₄ and extracted with 3×400 ml of ethyl acetate. The combinedorganic phases were concentrated in vacuo to a yellow solid 16 (mp49°-53° C., 103 g, 87% yield).

B. Preparation of 17, 19

2-p-Methoxybenzyl acetic acid 16 (3.92 g, 0.02 mol) was dissolved in 100ml of ether and cooled to -78° C. under a nitrogen atmosphere.Triethylamine was added (2.86 ml, 0.0205 mol) followed by pivaloylchloride (2.52 ml, 0.0205 mol). The mixture was warmed to 0° C. over 30min. and then stirred at 0° C. for 2 hr. to give mixed anhydride 17. Thesolution was then cooled to -78° C. (SOLUTION A).

In a separate flask the (S) phenylalanine derived oxazolidinone 18 (3.45g, 0.0195 mol) was dissolved in 30 ml of tetrahydrofuran and cooled to-78° C. under a nitrogen atmosphere. n-Butyllithium (14.3 ml of a 1.36Msolution in hexane) was added via cannula, and then was stirred for 15min. at -78° C. (SOLUTION B).

Solution B was then added, via cannula, to solution A at -78° C. Theresulting mixture was stirred 15 min. at -78° C., warmed to 0° C. over30 min., and then stirred for 1 hour at 0° C. Sixty ml of water was thenadded and the mixture was extracted with 3×50 ml of methylene chloride.The combined organic extracts were washed with 50 ml of saturated sodiumbicarbonate solution and 50 ml of saturated sodium chloride solution andwere dried over sodium sulfate. Concentration in vacuo and flashchromatography with silica gel (elution with 3:1, hexanes:ethyl acetate)gave the desired p-methoxybenzyl acetate derived oxazolidinone imide 19(5.51 g).

EXAMPLE 13(2S,3R,4R,6S,7R,8S,10S,14R,15S,17S,18R,19S,1'R,3'R-4'R)-E,E-2-(p-Methoxybenzyloxy)-3-hydroxy-4,10,12-18,20-pentamethyl-6,8-dimethoxy-7,15-bis-t-butyldimethylsilyloxy-14-(prop-2'-en-1'-yl)-17-triisopropylsilyloxy-19-((N-t-butylcarboyloxy-L-prolinoyloxy)-21-(3'-methoxy-4'-triisopropylsilylyoxycyclohexyl)-12,20-dienoicacid derived oxozolidin-2'-one imide 20. ##STR29##

p-Methoxybenzylacetimide 19 (286 mg, 3.0 equiv.) was dissolved in 6.3 mlof anhydzous toluene at -50° C. under a nitrogen atmosphere.Triethylamine (0.150 mL, 4.0 equiv) and dibutylboron triflate (0.195 ml,2.9 equiv) were added and the mixture was stirred for 1.5 hr. Aldehyde15 (361 mg, 0.262 mmol) in 3.6 ml of toluene was added and the mixturewas stirred for 1.0 hr at -50° C. The mixture was warmed to -30° C. andstirred for an additional 1.5 hr. Tlc analysis (3:1, hexanes:ethylacetate) showed the reaction to be complete at this time. The reactionmixture was quenched by the addition of 8.0 ml of saturated sodiumbicarbonate solution and then partitioned between 40 ml of aqueoussodium bicarbonate solutions and 75 ml of ethyl acetate. The layers wereseparated and the aqueous phase was extracted with 2×75 ml of ethylacetate. The combined organic layers were washed with 20 ml of saturatedsodium chloride solution and dried over sodium sulfate.

Concentration in vacuo gave 0.805 g of a crude oil. The product waspurified by column chromatography on 70 g of silica gel eluting withhexanes:ethyl acetate (3:1) to give imide 20 (0.385 g, 85%). Thematerial was homogeneous by ¹ H and ¹³ C NMR.

EXAMPLE 14(2S,3R,4R,6S,7R,8S,10S,14R,15S,17S,18R,19S,1'R,3'R-4'R)-E,E-2-(4'methoxybenzyloxy)-3-hydroxy-4,10,12-18,20-pentamethyl-6,8-dimethoxy-7,15-bis-t-butyldimethylsilyloxy-14-(prop-2'-en-1'-yl)-17-triisopropylsilyloxy-19-(N-t-butylcarboyloxy-L-prolinoyloxy)-21-(3'-methoxy-4'-triisopropylsilylyoxycyclohexyl)-12,20-dienoicacid, 21. ##STR30##

Imide 20 (1.191 g) was dissolved in 6.0 ml of tetrahydrofuran and 1.35ml of water and cooled to 0° C. Aqueous 30% hydrogen peroxide solution(0.560 ml, 8 equiv) and lithium hydroxide monohydrate (58 mg, 1.38 mmol)were added. The mixture was stirred at 0° C. for 1.5 hr. and thenconcentrated in vacuo to remove the tetrahydrofuran. The reaction wasquenched at 0° C. by addition of 7.3 ml of 10% aqueous sodium bisulfatesolution. The mixture was diluted with 20 ml of hexanes and the pH wasadjusted to 3.0-3.5 with 0.5N sodium bisulfate solution. The mixture wasextracted with 3×50 ml of hexanes and the combined organic layers werewashed with 20 ml of water and dried over sodium sulfate. Concentrationin vacuo gave 1.081g, quantitative yield, of the desired carboxylic acid21.

EXAMPLE 15 (2S,3R,4R,6S,7R,8S,10S,14R,15S,17S,18R,19S,1'R,3'R,-4'R)E,E-2-(4'methoxybenzyloxy)-3-triethylsilyloxy4,10,12,18,20-pentamethyl-6,8-dimethoxy-7,15-bis-t-butyldimethylsilyloxy-14-(prop-2'-en-1'-yl)-17-triisopropylsilyloxy-19-(L-prolinoyloxy)-21-(3'-methoxy-4'-triisopropylsilylyoxycyclohexyl)-12,20-dienoicacid, 22 ##STR31##

Acid 21 (200 mg) was dissolved in 7.0 ml of methylene chloride at 0° C.and 2,6-lutidine (6.0 equiv) was added. Triethylsilyltriflate (5.5equiv) was added and the mixture was stirred at -20° C. for 30 minutes.The reaction mixture was diluted with 5 ml of distilled water andextracted with 3×25 ml of hexane. The hexane portions were combined,dried over sodium sulfate and concentrated in vacuo (bath temperaturewas maintained at 15° C. or less). The concentrate was then dissolved inmethylene chloride and passed through a column containing 83.5 g ofsilica gel. Column elution was with 700 ml of methylene chloride; 930 mlof 1% methanol/methylene chloride; 700 ml 4% methanol/methylenechloride; and 1000 ml 8% methanol methylene chloride. The column richcuts were combined and concentrated to give 154 mg of the amino acid 22(76%).

EXAMPLE 16 C.9 -(p-Methoxybenzyloxy)-C.10-triethylsilyloxy-C.14,C.22-bis-t-butyldimethylsilyloxy-C.24,C.32-bis-triisopropylsilyloxy-hexahydro-FK-525 (FK-numbering system) 23.##STR32##

2-Chloro-N-methylpyridinium iodide (60 mg, 2.0 equiv) was dissolved in150 ml of methylene chloride under a nitrogen atmosphere andtriethylamine (0.078 mL, 5 equiv) was added. Amino acid 22 (186.5 mg) in15 ml of anhydrous methylene chloride containing 0.046 ml oftriethylamine was added via syringe over a period of 1.5 hr at 25° C.The mixture was stirred at 25° C. for 13 hr. and then diluted with 20 mlof water. The layers were separated and the aqueous layer was extractedwith 2×25 ml of methylene chloride. The organic portions were combineddried over sodium sulfate and concentrated in vacuo to give an oil. Thecrude oil was purified by column chromatography on silica gel (38 g,elution with hexanes/ethyl acetate 15:1). The rich cuts were combinedand concentrated to give the desired macrocycle 23 (130 mg, 70% yield).Macrocycle 23 was characterized by ¹ H and .sup. 13 C NMR.

EXAMPLE 17 C.9,C.10-Dihydroxy-C.14,C.22-bis-t-butyldimethylsilyloxy-C.24,C.32-bis-(triisopropylsilyloxy)-FK-525.24 ##STR33##

Macrocycle 23 (526 mg, 0.335 mmol) was dissolved in 3 ml of methylenechloride containing 0.18 ml of water and the mixture was stirred at 25°C. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (380 mg, 1.6 mmol) wasadded and the mixture was stirred at 25° C. for 5 hr. The crude mixturewas then chromatographed on 42 g of silica gel (400 mesh, eluting with200 ml methylene chloride; 320 ml 15:1 hexanes/ethyl acetate; 280 ml 6:1hexanes/ethyl acetate). The rich cuts were combined and concentrated invacuo to give the alcohol which was dissolved in 5.3 ml oftetrahydrofuran containing 0.6 ml of water and 0.086 ml oftrifluoracetic acid. The mixture was then stirred at 25° C. for 3.5 hr.The mixture was diluted with 10 ml of saturated sodium bicarbonatesolution and extracted with 3×25 ml of ethyl acetate. The combinedorganic phases were dried over sodium sulfate, concentrated in vacuo,and chromatographed on silica gel) elution with 6:1 hexanes:ethylacetate) to give 358 mg of diol 24 (80% yield). Microanalysis calcd forC₇₃ H₁₄₁ N₁₂ Si₄ : C, 65.567; H, 10.627; N, 1.047 Found C, 65.67; H,10.43; N=1.05. Rotation [α]²⁵ =56.5°, C=1.73 in methylene chloride.

EXAMPLE 18 C.14, C.22-bis-t-butyldimethylsilyloxy-C.24,C.32-bis(triisopropylsilyloxy)-FK-525, 25 ##STR34##

Oxalyl chloride (0.177 ml, 12 equivalents) was dissolved in 1.9 ml ofmethylene chloride and cooled to -78° C. under nitrogen. Dimethylsulfoxide (0.240 ml, 20 equiv) was added and the mixture was stirred for20 minutes. The dihydroxy macrocycle 24 (216 mg) dissolved in 2.0 ml ofmethylene chloride was added to the oxidant solution and the mixture wasstirred at -78° C. for 3 hr. Triethylamine (1.18 ml, 50 equiv) was addedand the mixture was warmed to -30° C. and stirred for 1 hr. The reactionwas quenched by the addition of 30 ml of 0.5N sodium bisulfate solutionand the mixture was extracted with 3×25 ml of ethyl acetate. The ethylacetate phases were combined, dried over sodium sulfate, concentrated invacuo and purified by chromatography on silica gel (eluting with 70 ml8:1 hexanes/ethyl acetate: 70 ml 6:1 hexanes/ethyl acetate) to give thedesired diketo macrocycle 25 as an oil. The product was resubjected tothe above reaction conditions to give 176 mg overall the desireddiketone 25 (77% overall yield). The diketo macrocycle 25 washomogeneous by both ¹ H and ¹³ C NMR.

EXAMPLE 19 C.22-dihydro-FK-525, 26. ##STR35##

Diketo-macrocycle 25 (144.7 mg, 0.108 mmol) was dissolved in 7 ml ofacetonitrile at 0° C. and 7 drops of 50% aqueous hydrofluoric acid wasadded. The mixture was stirred at 0° C. for 8 hr. and then diluted with18 ml of saturated aqueous sodium bicarbonate solution. The mixture wasextracted with 4×20 ml of ethyl acetate and the organic layers werecombined, dried over sodium sulfate, and concentrated in vacuo to an oil(102 mg). The oil was purified by chromatography on silica gel (10 g,elution with 100 ml 1:2 hexanes/ethyl acetate and then 100 ml of ethylacetate) to give 66 mg of compound 26 (77%). The material washomogeneous by ¹ H and ¹³ C NMR. Rotation [α]D C=-29.95°, C=0.661 inchloroform, IR 3600, 3550-3200, 1745, 1735 and 1630 cm⁻¹.

EXAMPLE 20 C.24,C.32-bis-triethylsilyloxy-dihydro-FK-525, 27 ##STR36##

Analog 26 (8.9 mg) was dissolved in anhydrous pyridine at 0° C. under anitrogen atmosphere and triethylsilychloride (0.008 ml, 4.2 equivalents)was added. The mixture was stirred for 12 hr at 0° and then diluted with5 ml of saturated sodium bisulfate solution. The mixture was extractedwith 3×5 ml of ethyl acetate, the organic phases were combined, driedover sodium sulfate, and concentrated in vacuo. Chromatography of theresidual oil on silica gel (elution with 60 nml 5:1 hexanes/ethylacetate; 50 ml of 4:1 hexanes/ethyl acetate; 80 ml of 3:1 hexanes/ethylacetate) to give the bis-triethylsilyloxy compound 27 (10.2 mg). Thematerial was characterized by ¹ H and ¹³ C NMR.

EXAMPLE 21 C.24,C.32-bis-triethylsilyloxy-dihydro-FK-525, 28 ##STR37##

The C.22 alcohol 27 (39.5 mg) was dissolved in 2.0 ml of CH₂ Cl₂ undernitrogen and pyridine (0.010 ml, 3 equiv) was added followed byDess-Martin perioindane (27 mg, 1.5 equiv). The reaction mixture wasaged for 1.5 hours at 25° C. Thin layer chromatography (hexanes:ethylacetate 2/1) showed the absence of starting material at this time. Themixture was partitioned between 5 ml of methylene chloride and 10 ml ofsaturated sodium sulfate, and concentrated in vacuo to give 50 mg of acrude oil. The oil was purified by chromatography on silica gel (10 g,elution with 3:1 hexanes/ethyl acetate) to give 32.1 mg (81% yield) ofthe desired bis-triethylsilyl FK-525 ketone 28. This material washomogeneous by both ¹ H and ¹³ C NMR.

EXAMPLE 22 FK-525, 29 ##STR38##

The silyl derivative 28 (32 mg) was dissolved in 2.0 ml of acetonitrileand cooled to 0° C. One drop of 50% aqueous hydrofluoric acid was addedand the mixture was aged at 0° C. for 1 hr. The mixture was diluted with5 ml of saturated aqueous sodium bicarbonate solution and extracted with5×20 ml of ethyl acetate. The organic phases were combined, washed with10 ml of saturated sodium chloride solution, dried over sodium sulfate,and concentrated in vacuo to give 14.2 mg of crude product. Thismaterial was purified by chromatography on silica gel (5 g, elution with50 ml of 1:2 hexanes/ethyl acetate and then 50 ml of ethyl acetate) togive 22.2 mg of the FK-525 29 (89% yield).

IR (CHCl₃): λ_(max) 3600, 3500-3200(vb), 1745, 1735, 1695, 1630 cm⁻¹

¹³ C NMR (CDCl₃, 32 mg/mL, major rotamer only, 75.5 MHz): δ 213.1,187.9, 168.8, 162.5, 140.4, 135.4, 132.1, 129.8, 122.1, 116.6, 99.0,84.1, 78.4, 76.5, 73.6, 73.5, 71.2, 69.0, 59.9, 57.7, 56.5, 56.2, 53.2,48.8, 48.5, 44.0, 41.0, 36.1, 35.4, 34.8, 34.7, 32.9, 32.6, 31.1, 30.6,28.4, 25.7, 25.4, 18.7, 16.1, 15.6, 14.0, 9.6.

[α]²⁵ =-98.5, C=0.195, CHCl₃.

What is claimed is:
 1. A compound of the formula: ##STR39##